Recent breakthroughs in the development of induced pluripotent stem cells have generated much interest in the therapeutic potential of stem cells in regenerative medicine. Pioneering work by Yamanaka and colleagues identified a transcription factor quartet (4F), Oct4, Sox2, Klf4 and c-Myc, that enables reprogramming of somatic cells to a pluripotent state (1, 2). The induced pluripotent stem cells (iPSCs) closely resemble embryonic stem cells (ESCs) in gene expression, epigenetic signature, and functional pluripotency. The simplicity of this reprogramming approach has opened up tremendous opportunities to generate patient-specific cells for disease modeling and therapeutic applications.
Two issues appear to limit the application of iPSCs, the low efficiency of reprogramming and the integration of transgenes into the somatic genome (3). The low efficiency and slow kinetics of reprogramming methods to generate iPSCs impose major limitations on their biomedical applications and continue to present a problem for ultimate therapeutic applications of iPSCs. There is thus a need for more efficient procedures for iPSC generation, and one approach is the use of small molecule chemicals to reprogram somatic cells with improved efficiency and kinetics.
Substantial effort has been made toward identifying chemical compounds that can enhance the efficiency of reprogramming (4-14). Several small molecules that are known to remodel chromatin and affect epigenetic control are being investigated actively for their effect on reprogramming. It has been shown that DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, and histone demethylase inhibitors can improve reprogramming efficiency (4, 5, 10, 11, 13). These compounds may act by reducing the epigenetic barriers to reprogramming, and they may potentially improve the efficiency and quality of the derived iPSCs (13). Molecules that act on known signaling pathways involved in ESC self-renewal and pluripotency, including Wnt, TGFβ, and MEK, have also been shown to enhance reprogramming efficiency (8, 9, 11, 15). More recently, retinoic acid receptor (RAR) agonists, vitamin C and lithium have been reported to enhance reprogramming efficiency as well (12, 16, 17).
Oct4 is a key regulator for ESC pluripotency. Reduced expression of Oct4 results in differentiation of ESCs into trophectodermal cells, while overexpression of Oct4 leads to differentiation of ESCs along the mesodermal and primitive endodermal lineages (18). Since the original report of induced reprogramming using the transcription factor quartet, Oct4, Sox2, Klf4, and c-Myc, the combination of factors used to generate iPSCs have been much studied (4-13, 15, 16, 19-32). However, Oct4 remains as the key, required component of the reprogramming cocktail, not replaceable by other factors, except the nuclear receptors NR5a1, NR5a2, and the combination of microRNAs miR-200c, miR-302s and miR-369s (23, 33, 34). Using neural stem cells that endogenously express Sox2, Klf4, and c-Myc, Oct4 was shown to be sufficient by itself to induce pluripotency (35, 36). The central role of Oct4 in the reprogramming process prompted us to ask whether Oct4-activating compounds may enhance reprogramming efficiency, thus improving iPSC technology.
In this study, high-throughput screening of small molecule libraries was performed to identify Oct4 promoter-activating compounds using a human Oct4 promoter-driven luciferase reporter. The identified compounds were characterized for their ability to enhance reprogramming efficiency and accelerate the reprogramming process. The derived iPSCs were characterized for their gene expression, epigenetic profile, and pluripotency. Disclosed herein, inter alia, are solutions to these and other problems in the art.